U.S. patent number 8,026,088 [Application Number 12/085,884] was granted by the patent office on 2011-09-27 for angiogenic tyrosyl trna synthetase compositions and methods.
This patent grant is currently assigned to The Scripps Research Institute. Invention is credited to Xiang-Lei Yang.
United States Patent |
8,026,088 |
Yang |
September 27, 2011 |
**Please see images for:
( Certificate of Correction ) ** |
Angiogenic tyrosyl tRNA synthetase compositions and methods
Abstract
The present invention provides an isolated tyrosyl tRNA
synthetase (TyrRS) polypeptide variant which comprises (a) a
Rossmann fold region or a portion thereof, preferably including an
5 coil; and (b) an anticodon recognition domain or portion thereof,
preferably including an 14 coil. Preferably, the 5 coil and the 14
coil have a greater spatial separation in the tertiary structure of
the variant compared to the corresponding spatial separation in
native human TyrRS. The variant preferably comprises an amino acid
residue sequence identity of at least about 50% compared to the
amino acid residue sequence of human TyrRS (SEQ ID NO: 3), includes
at least one non-conservative amino acid residue sequence of human
TyrRS, and preferably presents an exposed ELR motif in the 5 coil
on an external portion of the tertiary structure of the
polypeptide. A preferred TyrRS protein variant comprises the amino
acid residue sequence of SEQ ID NO: 4 or a portion thereof. The
proteins and protein fragments of the invention are angiogenic and
are useful for stimulating angiogenesis in mammalian tissues.
Inventors: |
Yang; Xiang-Lei (San Diego,
CA) |
Assignee: |
The Scripps Research Institute
(La Jolla, CA)
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Family
ID: |
38092855 |
Appl.
No.: |
12/085,884 |
Filed: |
December 1, 2006 |
PCT
Filed: |
December 01, 2006 |
PCT No.: |
PCT/US2006/046106 |
371(c)(1),(2),(4) Date: |
June 02, 2008 |
PCT
Pub. No.: |
WO2007/064941 |
PCT
Pub. Date: |
June 07, 2007 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090110672 A1 |
Apr 30, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60741580 |
Dec 2, 2005 |
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Current U.S.
Class: |
435/183;
536/23.2; 435/69.1; 435/320.1; 435/252.3 |
Current CPC
Class: |
A61P
9/00 (20180101); A61P 17/02 (20180101); A61P
29/00 (20180101); A61P 19/02 (20180101); A61P
43/00 (20180101); C12N 9/93 (20130101) |
Current International
Class: |
C12P
21/06 (20060101); C12N 9/00 (20060101); C12N
1/20 (20060101); C12N 15/00 (20060101); C07H
21/04 (20060101) |
Foreign Patent Documents
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WO 01/75078 |
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Oct 2001 |
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WO |
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WO 03/080648 |
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Oct 2003 |
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WO |
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Other References
Accession Q9VV60. Published May 1, 2000. cited by examiner .
Accession Q9VV60, published May 1, 2000. cited by examiner .
Yang et al. Trends Biochem Sci. May 2004;29(5):250-6. cited by
examiner.
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Primary Examiner: Fronda; Christian
Attorney, Agent or Firm: Olson & Cepuritis, Ltd.
Government Interests
STATEMENT OF GOVERNMENT INTEREST
A portion of the work described herein was supported by grant
number CA 92577 from the National Institutes of Health. The United
States Government has certain rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
for Patent Ser. No. 60/741,580, filed on Dec. 2, 2005, which is
incorporated herein by reference.
Claims
What is claimed is:
1. An isolated tyrosyl tRNA synthetase (TyrRS) polypeptide variant
comprising the amino acid residue sequence of SEQ ID NO: 4 or a
fragment thereof, wherein the fragment of SEQ ID NO: 4 comprises at
least all of residues 1 through 364 of SEQ ID NO: 4, and wherein
residue X at position 341 of SEQ ID NO: 4 is selected from the
group consisting of a glycine residue, an alanine residue, a valine
residue, a leucine residue, an isoleucine residue, a methionine
residue, and a proline residue.
2. The TyrRS polypeptide variant of claim 1 wherein residue X at
position 341 of SEQ ID NO: 4 is an amino acid residue selected from
the group consisting of a glycine residue, an alanine residue, and
a proline residue.
3. The TyrRS polypeptide variant of claim 1 wherein residue X at
position 341 of SEQ ID NO: 4 is an alanine residue.
4. An isolated tyrosyl tRNA synthetase (TyrRS) polypeptide variant
consisting of the amino acid residue sequence of SEQ ID NO: 4,
wherein residue X at position 341 of SEQ ID NO: 4 is selected from
the group consisting of a glycine residue, an alanine residue, a
phenylalanine residue, a valine residue, a leucine residue, an
isoleucine residue, a methionine residue, and a proline
residue.
5. The TyrRS polypeptide variant of claim 4 wherein residue X at
position 341 of SEQ ID NO: 4 is selected from the group consisting
of a glycine residue, an alanine residue, and a proline
residue.
6. The TyrRS polypeptide variant of claim 4 wherein residue X at
position 341 of SEQ ID NO: 4 is an alanine residue.
Description
FIELD OF THE INVENTION
This invention relates to angiogenic tyrosyl-tRNA synthetase
(TyrRS) compositions. More particularly this invention relates to
angiogenic TyrRS protein variants and angiogenic fragments thereof,
and to methods of stimulating angiogenesis therewith.
BACKGROUND OF THE INVENTION
Aminoacyl-tRNA synthetases are essential enzymes that catalyze the
addition of amino acids to their cognate tRNAs as a first step in
protein synthesis. These essential enzymes are separated into two
classes based on the presence of unique sequence motifs and overall
structure of their catalytic domains. Class I synthetases have two
highly conserved amino acid motifs, i.e., the sequences HIGH (SEQ
ID NO: 1) and KMSKS (SEQ ID NO: 2), within the catalytic domains of
its ten members. In contrast, the Class II synthetases have three
highly degenerate sequence motifs, referred to as motifs 1-3. Over
the past twenty years, several additional roles for tRNA
synthetases were discovered, including RNA splicing, nuclear export
and regulation of gene transcription. These additional functions
have been acquired during the long evolution of these ancient
enzymes. Many of these added functions are a result of appended
domains, which have been fused to the core synthetase sequences. In
higher eucaryotes, the appended domains of two synthetases have
been demonstrated to have functional biological roles unrelated to
the function of the core enzyme. For instance, fragments of two
human tRNA synthetases, i.e., tyrosyl-tRNA synthetase (TyrRS) and
tryptophanyl-tRNA synthetase (TrpRS), have been demonstrated to
have cytokine-like activities. Two related fragments of human
TrpRS, known as mini-TrpRS and T2-TrpRS, are negative regulators of
angiogenesis. Human TyrRS (SEQ ID NO: 3) can be readily separated
into two active fragments. A C-terminal appended domain fragment
has activity similar to the pro-inflammatory cytokine
endothelial-monocyte-activating polypeptide II (EMAP II), while an
N-terminal fragment (mini-TyrRS, residues 1-364 of SEQ ID NO: 3)
induces angiogenesis.
Angiogenesis is a tightly regulated process in which a careful
balance between pro-angiogenic and anti-angiogenic factors must be
maintained. Disruption of this balance, leading to excessive or
insufficient growth of blood vessels, is associated with diseases
such as age-related macular degeneration, rheumatoid arthritis,
delayed wound healing, as well as many other conditions. Regulation
is controlled through a variety of processes including
transcriptional and translational control, post-translational
modifications and processing of the ligand. Other proangiogenic
cytokines, including tumor necrosis factor-alpha and hepatocyte
growth factor, are generated by proteolytic cleavage of precursor
proteins. Similarly, cleavage by proteases releases active cytokine
fragments from human TrpRS and TyrRS.
The TrpRS and TyrRS enzymes of higher eukaryotes are composed of a
core catalytic region that includes a Rossmann fold having a number
of alpha coils interspersed with beta sheet segments. The Rossmann
fold catalytic domain of human TyrRS (residues 1 through 230 of SEQ
ID NO: 3) includes a hydrogen bond tether between the .alpha.5 coil
of the Rossmann fold domain and the .alpha.14 coil of the anticodon
recognition domain (see FIG. 3, Bottom Panel, and FIG. 4, Top
Panel). The tether partially blocks the Glu-Leu-Arg (ELR) motif in
the .alpha.5 coil of the active site domain of the protein.
The catalytic domain of human TyrRS and TrpRS are each homologous
to the catalytic domains of their respective corresponding
bacterial and lower eukaryotic enzymes, appended with a C-terminal
or N-terminal extension, respectively. The C-terminal extension of
human TyrRS shares about 51% sequence identity to the
pro-inflammatory cytokine EMAP II. In each case, the full-length
enzymes are inactive as cytokines, though functional as
synthetases. When the extensions unique to higher eukaryotes are
removed, the enzymes become active cytokines capable of controlling
angiogenesis.
It has now been discovered that opening the separation between the
.alpha.5 coil of the catalytic Rossmann fold domain and the
.alpha.14 coil of the anticodon recognition domain relative to the
separation of these coils in native human TyrRS renders the protein
angiogenic. The present invention provides TyrRS protein variants
and fragments thereof, which are useful for stimulating
angiogenesis in mammalian tissues.
SUMMARY OF THE INVENTION
The present invention provides a biologically active TyrRS
polypeptide variant, and angiogenic fragments thereof (collectively
referred to herein as "TyrRS polypeptide variants"), which are
suitable for stimulating angiogenesis in mammalian tissues. The
isolated tyrosyl tRNA synthetase (TyrRS) polypeptide variants of
the invention comprise a Rossmann fold domain or a portion thereof;
an anticodon recognition domain or a portion thereof; and include
at least one non-conservative amino acid residue substitution
relative to the amino acid residue sequence of human TyrRS (SEQ ID
NO: 3). The variants exhibit an angiogenic activity that is greater
than the angiogenic activity of native human TyrRS. Preferably, the
TyrRS polypeptide variants of the invention have an amino acid
residue sequence identity of at least 50% compared to the amino
acid residue sequence of human TyrRS (SEQ ID NO: 3), more
preferably at least 80% sequence identity, most preferably at least
about 95% sequence identity compared to SEQ ID NO: 3. Preferably,
the TyrRS polypeptide variants include a non-conservative amino
acid residue substitution of an amino acid residue at one or more
of positions corresponding to positions 46, 340, and 341 of SEQ ID
NO: 3.
In a preferred embodiment, the TyrRS polypeptide variant of the
invention comprises a Rossmann fold region or a portion thereof,
which includes an .alpha.5 coil, as well as an anticodon
recognition domain or a portion thereof that includes an .alpha.14
coil. The TyrRS polypeptide variant has an amino acid residue
sequence that has a sequence identity of at least about 50%
compared to the amino acid residue sequence of native human TyrRS
(SEQ ID NO: 3, FIG. 1). The variant includes at least one
non-conservative amino acid residue substitution relative to the
amino acid residue sequence of human TyrRS, which opens up the
separation between the .alpha.5 coil and the .alpha.14 coil,
relative to the separation of the .alpha.5 coil and the .alpha.14
coil in native human TyrRS. Preferably, the .alpha.14 coil is
separated by at least about 6 Angstroms from the .alpha.5 coil in
the tertiary structure of the variant, as determined by the spatial
separation between the alpha-carbon of any amino acid residue of
the .alpha.14 coil and the alpha-carbon of any amino acid residue
of the .alpha.5 coil. The variant preferably is free from hydrogen
bonds between the .alpha.5 coil and the .alpha.14 coil.
In another preferred embodiment of the TyrRS polypeptide variant of
the invention the .alpha.5 coil includes an ELR motif, and the
.alpha.14 coil is spaced at least about 6 Angstroms from the ELR
motif of the .alpha.5 coil in the tertiary structure of the
variant, as determined by the spatial separation between the
alpha-carbon of any amino acid residue of the .alpha.14 coil and
the alpha-carbon of any amino acid residue of the ELR motif of the
.alpha.5 coil. The variant presents an exposed ELR motif on an
external portion of the polypeptide tertiary structure. The TyrRS
polypeptide variant has an amino acid residue sequence identity of
at least about 50% compared to the amino acid residue sequence of
human TyrRS (SEQ ID NO: 3, FIG. 1), and includes at least one
non-conservative amino acid residue substitution relative to the
amino acid residue sequence of human TyrRS, which precludes
formation of a hydrogen bond tether between the ELR motif of the
.alpha.5 coil and the amino acid residues of the .alpha.14 coil of
the TyrRS polypeptide variant, or which otherwise results in
exposure of the ELR motif in the tertiary structure of the
variant.
In yet another preferred embodiment, the TyrRS polypeptide variant
includes a non-conservative amino acid residue substitution at an
amino acid residue corresponding to one or more of positions 46,
340, and 341 of SEQ ID NO: 3. A particularly preferred substitution
is replacement of the amino acid corresponding to the tyrosine at
position 341 of SEQ ID NO: 3 with an amino acid residue having an
aliphatic side chain, preferably a non-polar aliphatic side chain,
such as an alanine residue.
The TyrRS variants of the invention are suitable for stimulating
angiogenesis in mammalian (e.g., human) tissues.
The present invention also provides methods of stimulating
angiogenesis and endothelial cell migration in a tissue of a mammal
by contacting the tissue with a TyrRS polypeptide variant of the
invention, as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid residue sequence of human TyrRS (SEQ ID
NO: 3).
FIG. 2 shows the amino acid residue sequence of a preferred human
TyrRS variant of the invention (SEQ ID NO: 4).
FIG. 3 (Top Panel) shows a schematic diagram of the wild-type human
TyrRS sequence, which is composed of 3 domains: the Rossmann fold
catalytic domain (yellow, residues 1-230), the anticodon
recognition domain (green, residues 231-364) and the C-terminal
domain (purple, residues 364-528). The first two domains form the
active core enzyme, which is called mini TyrRS (residues 1-364).
The Bottom Panel shows a dimeric model of human TyrRS based on
crystal structures of both mini TyrRS and the C-domain, which were
experimentally determined.
FIG. 4 (Top Panel) shows details of the interaction of the ELR
region with other residues, including Y341. The Bottom Panel shows
a partial sequence alignment of TyrRS from a range of organisms in
which residues corresponding to three regions of human TyrRS are
aligned, i.e., residues 39-55 (Region 1), residues 87-97 (Region
2), and residues 337-346 (Region 3). Conserved residues involved in
the domain contacts are highlighted. Row 1 includes amino acid
residues of human TyrRS, in which Region 1 corresponds to SEQ ID
NO: 5, Region 2 corresponds to SEQ ID NO: 6, and Region 3
corresponds to SEQ ID NO: 7. Row 2 shows alignment of murine TyrRS,
in which Region 1 corresponds to SEQ ID NO: 5, Region 2 corresponds
to SEQ ID NO: 6, and Region 3 corresponds to SEQ ID NO: 7, as in
human TyrRS. Row 3 shows alignment of bovine TyrRS in which Region
1 corresponds to SEQ ID NO: 5, Region 2 corresponds to SEQ ID NO:
8, and Region 3 corresponds to SEQ ID NO: 9. Row 4 shows alignment
of Drosophila melanogaster TyrRS in which Region 1 corresponds to
SEQ ID NO: 5, Region 2 corresponds to SEQ ID NO: 10, and Region 3
corresponds to SEQ ID NO: 11. Row 5 shows alignment of
Caenorhabditis elegans TyrRS in which Region 1 corresponds to SEQ
ID NO: 12, Region 2 corresponds to SEQ ID NO: 13, and Region 3
corresponds to SEQ ID NO: 14. Row 6 shows alignment of
Saccharomyces cerevisiae TyrRS in which Region 1 corresponds to SEQ
ID NO: 15, Region 2 corresponds to SEQ ID NO: 16, and Region 3
corresponds to SEQ ID NO: 17. Row 7 shows alignment of
Saccharomyces pombe TyrRS in which Region 1 corresponds to SEQ ID
NO: 18, Region 2 corresponds to SEQ ID NO: 19, and Region 3
corresponds to SEQ ID NO: 20. Row 8 shows alignment of Escherichia
coli TyrRS in which Region 1 corresponds to SEQ ID NO: 21, Region 2
corresponds to SEQ ID NO: 22, and Region 3 corresponds to SEQ ID
NO: 23. Row 9 shows alignment of Bacillus subtilis TyrRS in which
Region 1 corresponds to SEQ ID NO: 24, Region 2 corresponds to SEQ
ID NO: 25, and Region 3 corresponds to SEQ ID NO: 26.
FIG. 5 shows an X-ray scattering distribution for human TyrRS and
TyrRS-Y341A. The electron pair distance distribution functions were
calculated for human TyrRS and the Y341A mutation of TyrRS using
their measured small angel x-ray scattering (SAXS) curves. Radius
of Gyration (R.sub.g) and Maximum distance (D.sub.max) were
calculated for both molecules. Y341A has an R.sub.g that is about 4
Angstroms larger, and a D.sub.max that is about 20 angstroms
larger, than those of wild-type TyrRS.
FIG. 6 schematically illustrates the opening effect of the Y341A
mutation on the overall tertiary structure of human TyrRS, which
exposes the ELR motif. The Y341A mutation was demonstrated by SAXS
analysis and protease digestion studies to open up the TyrRS
structure, as shown.
FIG. 7 illustrates the activity of TyrRS-Y341A in the mouse
Matrigel angiogenesis model. Mice received a 400 .mu.L subcutaneous
injection of growth factor-reduced Matrigel alone or mixed with 250
nM TyrRS proteins. Human VEGF.sub.165 (20 nM) was used as a
positive control. Following a five-day incubation, mice were
intravenously injected with fluorescein-labeled endothelial binding
lectin Griffonia (Bandeiraea) Sinplicifolia I, isolectin B4. The
plugs were excised, solubilized, and assessed for fluorescein
content by spectrometry. The opened-up TyrRS Y341A variant was
observed to have increased angiogenesis activity relative to
wild-type TyrRS.
FIG. 8 illustrates the activity of human TyrRS and TyrRS-Y341A in
the chick chorioallantoic membrane assay. Angiogenic activity of
TyrRS proteins was tested in vivo with the chick chorioallantoic
membrane assay. TyrRS variants (1 .mu.M), a positive control
consisting of a combination of VEGF and bFGF, as well as a negative
control consisting of buffer alone, were applied to the CAM in a
nylon mesh-imbedded collagen onplant.
FIG. 9 shows a graph of the percentage area (vertical axis) of the
remaining cells-free area compared to the area of the initial wound
in wounded endothelial cell cultures treated with TyrRS variants of
the invention and control treatments.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Amino acid residues in proteins have been classified in a variety
of ways, primarily based on the physico-chemical characteristics
imparted by the side chains of the amino acids. For example, one
common classification includes three categories of amino acids: (1)
hydrophobic (non-polar) amino acids, including glycine, alanine,
valine, phenylalanine, proline, methionine, isoleucine, and
leucine; (2) charged amino acids, including aspartic acid, glutamic
acid, lysine, and arginine; and (3) polar amino acids, including
serine, threonine, tyrosine, histidine, cysteine, asparagine,
glutamine, and tryptophan; glycine is sometimes included as its
own, forth category (see Chapter 1 of Branden and Tooze,
Introduction to Protein Structure, Second Edition, Garland
Publishing, Inc. 1998, pages 3-12, which is incorporated herein by
reference).
Amino acid residues can also be further categorized based on
whether their side chains are aliphatic or aromatic, small or
bulky, polar or non-polar, charged or non-charged, combinations
thereof, and the like. As used herein, non-polar, aliphatic amino
acids include glycine, alanine, valine, leucine, isoleucine,
methionine, and proline; non-charged, bulky amino acids include
tyrosine, tryptophan, phenylalanine, leucine, isoleucine, and
methionine; small, non-polar amino acids include glycine, alanine,
and proline.
As used herein and in the appended claims, the term
"non-conservative", when used in relation to amino acid residue
substitutions means the substitution of an amino acid residue in a
wild-type or natural protein with an amino acid residue of a
significantly different structural category, e.g., having a
significantly different polarity classification (i.e., non-polar
versus polar), size classification, charge classification, a
combination thereof, and the like. For example, for a
non-conservative substitution, a polar amino acid such as tyrosine
can be replaced by a non-polar amino acid, a relatively small
non-polar amino acid, and the like; while a small non-polar amino
acid such as glycine or alanine can be replaced by a bulky amino
acid, a charged amino acid, and the like.
An preferred isolated TyrRS polypeptide variant embodying the
present invention is suitable for stimulating angiogenesis in
mammalian tissues and comprises a Rossmann fold region or a portion
thereof, which includes an .alpha.5 coil, as well as an anticodon
recognition domain or a portion thereof that includes an .alpha.14
coil. The variant has a separation between the .alpha.5 coil and
the .alpha.14 coil in the variant, which is greater than the
separation between the .alpha.5 coil and the .alpha.14 coil in
native human TyrRS. Preferably, the .alpha.14 coil is separated
from the .alpha.5 coil by at least about 6 Angstroms in the
tertiary structure of the variant, as determined by the spatial
separation between the alpha-carbon of any amino acid residue of
the .alpha.14 coil and the alpha-carbon of any amino acid residue
of the .alpha.5 coil. The variant preferably is free from hydrogen
bonds between the .alpha.5 coil and the .alpha.14 coil. The TyrRS
polypeptide variant has an amino acid residue sequence identity of
at least about 50% compared to the amino acid residue sequence of
native human TyrRS (SEQ ID NO: 3, FIG. 1), more preferably at least
about 80%, most preferably at least about 95% sequence identity.
The variant includes at least one non-conservative amino acid
residue substitution relative to the amino acid residue sequence of
human TyrRS, which opens up the separation between the .alpha.5
coil and the .alpha.14 coil, relative to the separation of the
.alpha.5 coil and the .alpha.14 coil in native human TyrRS, and
confers angiogenic activity to the variant.
In another preferred embodiment, the TyrRS variant of the invention
comprises a Rossmann fold region or a portion thereof that presents
an exposed ELR motif on an external portion of the tertiary
structure of the polypeptide, and which has an amino acid residue
sequence identity of at least about 50% compared to the amino acid
residue sequence of human TyrRS (SEQ ID NO: 3), preferably at least
about 80%, more preferably about 95% sequence identity. The TyrRS
polypeptide variant includes at least one non-conservative amino
acid residue substitution relative to the amino acid residue
sequence of human TyrRS, which exposes the ELR motif of the
.alpha.5 coil of the catalytic Rossmann fold domain, thereby
rendering the polypeptide angiogenic. In the variants of the
invention, the ELR residues of the .alpha.5 coil preferably are
spaced at least about 6 Angstroms from and the residues of the
.alpha.14 coil each in the tertiary structure of the variant, as
determined by the spatial separation between the alpha-carbon of
any amino acid residue of the .alpha.14 coil and the alpha-carbon
of any amino acid residue of the ELR motif of the .alpha.5
coil.
Preferably, the at least one non-conservative amino acid residue
substitution is a substitution of an amino acid residue at one or
more of positions corresponding to amino acid residues 46, 340, and
341 of SEQ ID NO: 3. For example, the tyrosine at position 341 of
SEQ ID NO: 3 preferably is replaced by an amino acid residue having
a non-polar side chain (e.g., a non-polar, aliphatic amino acid,
such as glycine, alanine, valine, leucine, isoleucine, methionine,
and proline). The amino acid residue corresponding to the glycine
at position 46 and/or the alanine at position 340 of SEQ ID NO: 3
preferably is replaced by an amino acid residue having a bulky
non-polar side chain (e.g., a large non-charged, hydrophobic amino
acid such as tyrosine, tryptophan, phenylalanine, leucine,
isoleucine, or methionine). As used herein, a reference to an amino
acid in a TyrRS polypeptide variant "corresponding to" an amino
acid residue within a specified sequence, such as SEQ ID NO: 3,
means an amino acid at a position in the homologous sequence of the
TyrRS polypeptide variant, which aligns with (i.e., corresponds to)
the specified position in SEQ ID NO: 3 when the homologous sequence
is compared to the specified sequence. One of ordinary skill in the
protein arts will understand that the numbering of the amino acid
residue in the homologous sequence may be different from the
numbering in the specified sequence (e.g., SEQ ID NO: 3).
In yet another embodiment, an isolated TyrRS polypeptide variant of
the present invention comprises a Rossmann fold region or a portion
thereof that presents an exposed ELR motif on an external portion
of the protein or fragment and has an amino acid residue sequence
identity of at least about 50% compared to the amino acid residue
sequence of human TyrRS (SEQ ID NO: 3). Preferably, the amino acid
residue sequence of the variant has a sequence identity of at least
about 80% compared to SEQ ID NO: 3, more preferably about 95%, and
includes an amino acid residue having a non-polar side chain at the
position corresponding to position 341 of SEQ ID NO: 3. Preferably,
the amino acid residue has a non-polar, aliphatic side chain. For
example, the amino acid residue of the variant corresponding to
tyrosine residue 341 of human TyrRS can be a glycine residue, an
alanine residue, a phenylalanine residue, a valine residue, a
leucine residue, an isoleucine residue, a methionine residue, or a
proline residue. In some embodiments, the amino acid residue
corresponding to tyrosine 341 of human TyrRS is a glycine residue,
an alanine residue, or a proline residue, preferably an alanine
residue.
In another preferred embodiment, the isolated TyrRS polypeptide
variant comprises the amino acid residue sequence of SEQ ID NO: 4
(FIG. 2) or a fragment thereof, wherein residue X at position 341
of SEQ ID NO: 4 is a glycine residue, an alanine residue, a
phenylalanine residue, a valine residue, a leucine residue, an
isoleucine residue, a methionine residue, or a proline residue,
preferably a glycine, alanine, or proline residue, more preferably
an alanine residue. Preferably, the TyrRS polypeptide variant
comprises the entire N-terminal region (i.e., residues 1-364) of
SEQ ID NO: 4. Preferably, a fragment includes at least residues of
the .alpha.5 chain of the Rossmann fold region, i.e., residues
corresponding to positions 87 through 104 of SEQ ID NO: 4. More
preferably, the fragment encompasses the entire Rossmann fold
region (residues 1-230 of SEQ ID NO: 4).
A particularly preferred angiogenic TyrRS polypeptide variant of
the invention consists of the amino acid residue sequence of SEQ ID
NO: 4, wherein residue X at position 341 of SEQ ID NO: 4 is
selected from the group consisting of a glycine residue, an alanine
residue, a phenylalanine residue, a valine residue, a leucine
residue, an isoleucine residue, a methionine residue, and a proline
residue. More preferably, residue X at position 341 of SEQ ID NO: 4
is selected from the group consisting of a glycine residue, an
alanine residue, and a proline residue, most preferably an alanine
residue.
The present invention also provides a method of stimulating
angiogenesis in the tissue of a mammal. The method comprises
contacting the tissue with an angiogenic amount of a TyrRS
polypeptide variant of the invention as described in detail
herein.
In another aspect, the present invention provides a method of
stimulating endothelial cell migration in the tissue of a mammal.
The method comprises contacting the tissue with an endothelial
cells migration stimulating amount of a TyrRS polypeptide variant
of the invention as described in detail herein.
Methods and Procedures.
Plasmid Construction. TyrRS-Y341A and mini-TyrRS-Y341A plasmids
were constructed using the QuikChange site-directed mutagenesis kit
(Stratagene, La Jolla, Calif.). Templates were pET20b(+) (Novagen,
Masdison, Wis.) vectors containing the wild-type TyrRS and
mini-TyrRS, respectively. All proteins were expressed with a
C-terminal His-Tag to facilitate isolation of the polypeptide.
Synthetic oligonucleotides were purchased from Invitrogen
Corporation (Carlsbad, Calif.).
Protein Production and Endotoxin Removal. Recombinant polypeptides
were expressed in E. coli B121-CodonPlus (DE3)-RIL cells
(Stratagene, La Jolla, Calif.). Cells were grown to an OD.sub.600
of 0.8, and induced for 3 hours with 1 mM isopropyl
.beta.-D-thiogalactopyranoside (Roche, Basel, Switzerland). Cells
were then pelleted and resuspended in Buffer A (20 mM Tris-HCl, pH
7.9, 30 mM imidazole, 500 mM NaCl). Following lysis by sonication,
cell debris was separated by centrifugation at 74,000 g for 30
minutes. The His-tagged polypeptides were purified by Ni-NTA
affinity chromatography. Supernatant was loaded onto a Ni-NTA
affinity column (Qiagen, Valencia, Calif.), washed with 100 mL
Buffer B (Buffer A with 0.1% Triton-X114 (Sigma, St. Louis, Mo.))
and 150 mL Buffer A. Polypeptides were eluted by a linear gradient
of Buffer A and Buffer C (20 mM Tris-HCl, pH 7.9, 250 mM imidazole,
500 mM NaCl). Fractions containing >95% pure polypeptides were
pooled, concentrated and dialyzed into storage buffer (50%
phosphate buffered saline, PBS, pH 7.4, 50% glycerol, 2 mM
dithiothreitol, DTT). Protein concentration was determined by
Bradford assay using the Bio-Rad Protein Assay reagent (Bio-Rad,
Hercules, Calif.) with bovine serum albumin (BSA, Sigma, St. Louis,
Mo.) as a standard. Endotoxin concentration was determined using
the Limulus Amebocyte Lysate (LAL) assay (BioWhittaker,
Walkersville, Md.).
Small angel X-ray scattering. Small Angel X-ray scattering (SAXS)
measurements were conducted for the wild-type TyrRS and the
TyrRS-Y341A variant of the invention on beamline 4-2 at the
Stanford Synchrotron Radiation Laboratory (SSRL). X-ray scattering
curves were measured for samples at various concentrations (2-20
g/L). The x-ray wavelength (.lamda.) was 1.38 .ANG. and the
detector channel numbers were converted to the momentum transfer
Q=4.pi.*sin .theta./.lamda., where 2*.theta. is the scattering
angle. Two different sample-to-detector distances were used to
cover very small to medium scattering angles, i.e., 2.5 meters
covering a Q range of about 0.01-0.25 .ANG..sup.-1, and 0.5 meters
to cover a Q range of about 0.03-0.93 .ANG..sup.-1.
A polycarbonate cell with mica windows was filled with a sample
aliquot and held at 20.degree. C. throughout the measurement. A
MarCCD 165 detector was used throughout the data collection. A
typical set of data collection consisted of 24 two-dimensional
scattering images recorded in series for 10 seconds each. A series
of data were processed along with the matching buffer scattering
data, typically recorded either immediately after or before the
protein solution measurement. The data were scaled for the
integrated beam intensity, azimuthally averaged, inspected for
time-dependent changes, which are usually caused by radiation
damage, and statistically analyzed. About 1.5 times higher
statistical variations of the protein data over the variation of
the matching buffer data were allowed in the averaging. Any data
frame that showed a higher level of deviation with respect to the
first protein scattering data frame beyond that level was rejected.
The processed buffer scattering curves were subtracted from the
corresponding protein scattering curves after the above data
processing.
The scaling of small-angle and high-angle data were performed by
the PRIMUS software program (Konarev et al. 2003. "PRIMUS: a
Windows PC-based system for small-angle scattering data analysis",
J. Appl. Crystallogr. 36:1277-1282), which was also used to compute
R.sub.g and I(Q=0) by a Guinier plot in the Q*R.sub.g range of
0.45-1.3. The electron pair distance distribution function P(r) was
obtained by the indirect Fourier transfer of the experimental data
using the GNOM small-angle scattering data processing program of
Svergun et al. (available from the embl-hamburg(dot)de website),
initially with the small-angle data only to obtain accurate
estimate of the maximum distance (D.sub.max) P(r) was then
recomputed including the high-angle data with the specified
D.sub.max values above for model construction.
Protease digestion. Wild type TyrRS and TyrRS-Y341A were mixed with
plasmin or leukocyte elastase at a protein-to-protease ratio of
about 32 .mu.g to 1 .mu.g of plasmin, and 2500 .mu.g per unit for
the elastase. The mixtures were incubated at 20.degree. C. for
about 2 hours before the reactions were stopped by addition of
formic acid to a final concentration of 0.1%. The cleavage
fragments in the mixtures were analyzed by MALDI TOF mass
spectrometry and by N-terminal sequencing using Edman
degradation.
CAM Angiogenesis Assay. Chick chorioallantoic membrane (CAM)
angiogenesis assays were performed by the following procedure.
Fertilized complement fixation for avian leukosis virus
(COFAL)-negative eggs (Charles River Labs, Storrs, Conn.) were
incubated for 3.5 days at 38.degree. C./60% humidity. Eggs were
then opened and the embryos were transferred into sterile plastic
weigh boats. The embryos were covered and incubated at 37.5.degree.
C./90% humidity. After five days, collagen/mesh onplants containing
about 30 .mu.L of PBS, VEGF and bFGF (about 0.15 and 0.5 .mu.g,
respectively) or TyrRS polypeptides (1 .mu.M) were placed onto the
CAM membrane of the embryos, and incubated for an additional 66
hours. The upper mesh layers of the implants were examined under a
stereomicroscope and scored for the proportion of "boxes" (i.e.,
three dimensional regions defined by the mesh fibers), which
contain a blood vessel relative to the total number of boxes.
Murine Matrigel Angiogenesis Assay. Athymic wehi mice were
subcutaneously implanted with 400 .mu.L of growth-factor-depleted
Matri gel (Becton Dickinson) supplemented with PBS, 20 nM VEGF or
250 nM TyrRS, mini-TyrRS, TyrRS-Y341A or mini-TyrRS-Y341A. Five
days later, the mice were injected intravenously with
fluorescein-labeled endothelial binding lectin Griffonia
(Bandeiraea) Simplicifolia I, isolectin B4 (GSL-B4) (Vector
Laboratories, Burlingame, Calif.). Matrigel plugs were resected and
homogenized in radioimmuno-precipitation (RIPA) buffer (10 mM
sodium phosphate, pH 7.4, 150 mM sodium chloride, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Following
homogenization, the fluorescein content of each plug was quantified
by spectrophotometric analysis.
Endothelial Cell Migration Assay. Human umbilical vein endothelial
cells (HUVECs) (Cambrex) were plated at a density of about
3.times.10.sup.5 cells/well of a 6-well plate in EGM media
(Cambrex) containing 10% fetal bovine serum (FBS) and grown to
confluent monolayers. Cells were starved in media containing no FBS
for 16 hours and wounded across the well with a pipette tip. The
wounded layers were washed twice with serum-free media to remove
cell debris and the cells were allowed to migrate in the presence
and absence of TyrRS variants and control factors. Images of the
cell-free wound area were taken at 0 and 6 hours post wounding and
analyzed using an image analysis software (NIH ImageJ 1.33).
Endothelial cell migration was calculated as the percentage of the
remaining cell-free area compared to the area of the initial wound,
from which the % area closed for each condition was determined.
Amino Acid Activation. Amino acid activation was assayed by
ATP-pyrophosphate (PPi) exchange reaction. Reactions were performed
at 37.degree. C. in buffer containing Tris-HCl (100 mM, pH 7.8), KF
(10 mM), MgCl.sub.2 (2 mM), ATP (1 mM), BSA (0.1 mg/mL), NaPPi (2
mM, 130 cpm/nmole), beta-mercaptoethanol (5 mM), tyrosine (2 mM)
and 200 nM of the polypeptide.
Results and Discussion
Residue Y341 of TyrRS secured the structure around the ELR motif.
It is known that mini-TyrRS (i.e., amino acid residues 1-364 of
human TyrRS), but not full-length TyrRS, acts as a pro-angiogenic
cytokine. Pro-angiogenic activity is dependent upon the presence of
an ELR motif, which is also present in and required for activity of
CXC chemokines, such as IL-8. A schematic of the amino acid
sequence of TyrRS, which is comprised of 3 domains: the Rossmann
fold catalytic domain (yellow, residues 1-230), the anticodon
recognition domain (green, residues 231-364) and the C-terminal
domain (purple, residues 364-528) is shown in the Top Panel of FIG.
3. The first two domains form the active core enzyme, which is
called mini TyrRS (residues 1-364). The recently solved tertiary
structure of mini-TyrRS reveals a hydrogen-bonding network around
the ELR motif, which tethers the anticodon recognition and active
site domains of the protein (see FIG. 3, Bottom Panel). There are
two hydrogen bonding interactions between side chain of R93 of the
ELR motif and main chain A340 of the anticodon recognition domain,
which tether the .alpha.5 coil to the .alpha.14 coil. Additionally,
stacking interactions between the aromatic ring of Y341 and the
main chain G46, create a contact between these domains. Of these
four residues, R93, G46 and Y341 are conserved in all eukaryotic
TyrRS proteins identified so far. As shown in FIG. 3 (Bottom
Panel), the complementarity of the surface electrostatic potential
of each fragment provides evidence for the tether between the
.alpha.5 coil and the .alpha.14 coil. The ELR motif (on helix
.alpha.5) is masked by the C-domain, and is exposed when the
C-domain is removed. For this reason, mini TyrRS is active, whereas
the full-length TyrRS is inactive for angiogenesis. The Tyr341
residue (from the anticodon recognition domain on helix a 14)
interacts with the ELR, and plays an important role in tethering
C-domain to block the ELR. The A340 is also highly conserved, being
replaced only by another small amino acid, glycine, in S.
cerevisiae.
Two independent methods were used to demonstrate the
three-dimensional structure opening of TyrRS by the Y341A mutation.
The first method was Small Angel X-ray Scattering (SAXS). When the
wavelength of electromagnetic radiation is on the same length scale
as that of a sample particle, the particle will scatter the
radiation. Detection and analysis of this scattering pattern can
yield valuable information about the size, shape, and internal
structure of the particle. The SAXS technique is a powerful
technique to detect overall conformational change of macromolecules
(like proteins) in solution. From the scattering curves measured
with both the wild type human TyrRS and the Y341A mutation, the
electron pair distance distribution function for each sample was
calculated (FIG. 5). Values for the Radius of Gyration (R.sub.g)
and Maximum distance (D.sub.max) were subsequently derived from the
distribution functions, P(r).
FIG. 4 shows the hydrogen bond tether between R93 and Y341.
Disruption of the hydrogen bond tether opens up the tertiary
structure and exposes the ELR motif. Small angle X-ray scattering
studies on wild-type human TyrRS and a variant in which Y341 has
been replaced by an alanine (TyrRS-Y341A) provides direct evidence
for such opening (see FIG. 5). The X-ray scattering studies showed
that the electron pair distance distribution broadened in the
variant relative to the wild-type protein. TyrRS Y341A has an
R.sub.g of about 4 Angstroms larger than those of the wild type
TyrRS, and a D.sub.max about 20 Angstroms larger. This demonstrates
that TyrRS Y341A has a more open and extended conformation relative
to wild-type TyrRS.
Additional support for an open structure in TyrRS-Y341A relative to
wild-type human TyrRS was obtained from digestion studies using
plasmin and elastase. Proteases generally cleave proteins at
flexible loop regions, where there are no defined secondary
structures like alpha helices or beta strands. The presence of
structural opening to expose the ELR motif associated with the
Y341A mutation was confirmed by the additional cleavage fragments
that were observed as a result of additional cleavage site(s) on
the more relaxed, open structure. Using both plasmin and leukocyte
elastase, additional cleavage sites on the Y341A mutation were
observed, relative to the wild type TyrRS. The observed additional
cleavage sites were L333, K334 and A337, which are all located on
helix alpha 14 of the anticodon recognition domain, close to the
mutation site 341. This confirmed that in Y341A mutation, alpha 14
is relaxed and presents itself as a flexible loop, no longer
tethered to the ELR region. FIG. 6 schematically illustrates the
opening effect of the Y341A mutation on the overall tertiary
structure of human TyrRS.
The Y341A mutation conferred angiogenic activity onto the
full-length TyrRS. Y341 is an ideal target for mutation, given its
conservation among eucaryotes and its involvement in the tertiary
structural stability in the region around the ELR motif. This
residue was mutated to alanine in full-length TyrRS and mini-TyrRS
to determine the extent to which disruption of the tether alters
cytokine and enzymatic activities. Cytokine activity was tested in
the chick chorioallantoic membrane (CAM) and the Matrigel assays
for angiogenesis.
The inclusion of a Y341A mutation in mini-TyrRS produced no
observable effect on its activity in the CAM assay--angiogenesis
was promoted by both the normal polypeptide and the variant.
Full-length, wild-type human TyrRS is inactive in this assay.
However, mutation of Y341 of human TyrRS to an alanine (i.e.,
TyrRS-Y341A) converted this enzyme into a pro-angiogenic cytokine.
To confirm the results seen in the CAM assay, the polypeptides were
also tested in the mouse Matrigel plug model. In this assay, a
collagen plug containing PBS or other protein was injected
subcutaneously. When there is a pro-angiogenic factor within the
plug, blood vessels perfuse into the plug. Upon injection of a
fluorescent endothelial-cell binding lectin, the blood vessel
density in each plug can be quantified by spectrophotometric
analysis. In this model, VEGF and mini-TyrRS were both positive for
angiogenic activity. Wild-type TyrRS was inactive, with levels of
angiogenesis similar to PBS. In contrast, TyrRS-Y341A induced
angiogenesis. These data independently confirmed the CAM assay
results.
The TyrRS variant having the Y341A mutation caused a marked
increase in the vascularization of the area of the CAM membrane
containing the implant while neither the wild-type polypeptide or
PBS induced a response (polypeptides were applied at a
concentration of 1 .mu.M in a nylon mesh-imbedded collagen
implant).
FIG. 7 further characterizes the in vivo activity of the TyrRS
polypeptides in the mouse Matrigel plug assay. Mice were
administered a 400 .mu.L subcutaneous injection of growth
factor-reduced Matrigel alone or mixed with 250 nM protein.
Following a five-day incubation, the plugs are removed and assessed
for the presence of blood vessels. VEGF.sub.165 (20 nM) was used as
a positive control. As previously reported, mini-TyrRS promoted an
angiogenic response in this assay. The mutation of Y341 to alanine
in mini-TyrRS (i.e., mini-TyrRS-Y341A) did not affect this
response. However, when the Y341A mutation is made in the
full-length TyrRS, a 2-fold increase in angiogenic response was
noted as compared to the wild-type TyrRS.
The Y341A mutation inactivated the ability of TyrRS to activate
tyrosine. Amino acid activation was assessed by measuring the
tyrosine-dependent ATP-PPi exchange reaction. As shown in FIG. 8,
the Y341A point mutation knocks out the ability of the full-length
TyrRS, as well as the mini-TyrRS, to form the tyrosyl adenylate.
Thus, the structural elements that are involved in aminoacylation
of tRNA are different from those involved in cytokine function.
An endothelial cell migration assay was used to assess the ability
of the TyrRS variants of the invention to stimulate endothelial
cell migration, which is associated with wound healing. Human
umbilical vein endothelial cells (HUVECs) were plated in EGM media
containing 10% FBS and grown to confluent monolayers. The cells
were starved in media containing no FBS and then wounded across the
well with a pipette tip. The wounded layers were washed with
serum-free media to remove cell debris and the cells were allowed
to migrate in the presence and absence of TyrRS variants and
control factors. Images of the cell-free wound area were taken at 0
and 6 hours post wounding and analyzed using an image analysis
software (NIH ImageJ, version 1.33). Endothelial cell migration was
calculated as the percentage of the remaining cell-free area
compared to the area of the initial wound, from which the % area
closed for each condition was determined. FIG. 9 shows a graph of
the percentage (vertical axis) of the remaining cell-free area
compared to the area of the initial wound in wounded endothelial
cell cultures treated with TyrRS variants of the invention and
control treatments. A relatively smaller percentage of cell-free
area indicates improved endothelial cell migration and wound
healing. As shown in FIG. 9, the TyrRS variants of the invention
stimulated endothelial cell migration to an extent comparable to
miniTyrRS, and to a greater extent than wild-type TyrRS.
Numerous variations and modifications of the embodiments described
above may be effected without departing from the spirit and scope
of the novel features of the invention. No limitations with respect
to the specific embodiments illustrated herein are intended or
should be inferred.
SEQUENCE LISTINGS
1
2614PRTHomo sapiens 1His Ile Gly His 125PRTHomo sapiens 2Lys Met
Ser Lys Ser 1 53528PRTHomo sapiens 3Met Gly Asp Ala Pro Ser Pro Glu
Glu Lys Leu His Leu Ile Thr Arg 1 5 10 15Asn Leu Gln Glu Val Leu
Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys 20 25 30Glu Arg Glu Leu Lys
Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45His Val Ala Tyr
Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60Ala Gly Cys
Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu65 70 75 80Asp
Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90
95Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu
100 105 110Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser
Lys Glu 115 120 125Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val
Thr Gln His Asp 130 135 140Ser Lys Lys Ala Gly Ala Glu Val Val Lys
Gln Val Glu His Pro Leu145 150 155 160Leu Ser Gly Leu Leu Tyr Pro
Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175Leu Lys Val Asp Ala
Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190Thr Phe Ala
Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205His
Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215
220Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu
Asp225 230 235 240Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro
Gly Asn Val Glu 245 250 255Asn Asn Gly Val Leu Ser Phe Ile Lys His
Val Leu Phe Pro Leu Lys 260 265 270Ser Glu Phe Val Ile Leu Arg Asp
Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285Tyr Thr Ala Tyr Val Asp
Leu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300His Pro Gly Asp
Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu305 310 315 320Leu
Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330
335Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys
340 345 350Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser
Arg Leu 355 360 365Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys
His Pro Asp Ala 370 375 380Asp Ser Leu Tyr Val Glu Lys Ile Asp Val
Gly Glu Ala Glu Pro Arg385 390 395 400Thr Val Val Ser Gly Leu Val
Gln Phe Val Pro Lys Glu Glu Leu Gln 405 410 415Asp Arg Leu Val Val
Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg 420 425 430Gly Val Glu
Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile 435 440 445Asn
Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly 450 455
460Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu
Glu465 470 475 480Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln
Ala Asp Phe Lys 485 490 495Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys
Gln Thr Asn Phe Met Thr 500 505 510Lys Leu Gly Ser Ile Ser Cys Lys
Ser Leu Lys Gly Gly Asn Ile Ser 515 520 5254528PRTArtificial
SequenceVariant of human TyrRS 4Met Gly Asp Ala Pro Ser Pro Glu Glu
Lys Leu His Leu Ile Thr Arg 1 5 10 15Asn Leu Gln Glu Val Leu Gly
Glu Glu Lys Leu Lys Glu Ile Leu Lys 20 25 30Glu Arg Glu Leu Lys Ile
Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45His Val Ala Tyr Phe
Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60Ala Gly Cys Glu
Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu65 70 75 80Asp Asn
Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90 95Tyr
Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu 100 105
110Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu
115 120 125Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln
His Asp 130 135 140Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val
Glu His Pro Leu145 150 155 160Leu Ser Gly Leu Leu Tyr Pro Gly Leu
Gln Ala Leu Asp Glu Glu Tyr 165 170 175Leu Lys Val Asp Ala Gln Phe
Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190Thr Phe Ala Glu Lys
Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205His Leu Met
Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220Ser
Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp225 230
235 240Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val
Glu 245 250 255Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe
Pro Leu Lys 260 265 270Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp
Gly Gly Asn Lys Thr 275 280 285Tyr Thr Ala Tyr Val Asp Leu Glu Lys
Asp Phe Ala Ala Glu Val Val 290 295 300His Pro Gly Asp Leu Lys Asn
Ser Val Glu Val Ala Leu Asn Lys Leu305 310 315 320Leu Asp Pro Ile
Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335Ala Ser
Ala Ala Xaa Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345
350Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu
355 360 365Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro
Asp Ala 370 375 380Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu
Ala Glu Pro Arg385 390 395 400Thr Val Val Ser Gly Leu Val Gln Phe
Val Pro Lys Glu Glu Leu Gln 405 410 415Asp Arg Leu Val Val Val Leu
Cys Asn Leu Lys Pro Gln Lys Met Arg 420 425 430Gly Val Glu Ser Gln
Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile 435 440 445Asn Arg Gln
Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly 450 455 460Glu
His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu465 470
475 480Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe
Lys 485 490 495Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn
Phe Met Thr 500 505 510Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys
Gly Gly Asn Ile Ser 515 520 525517PRTHomo sapiens 5Tyr Trp Gly Thr
Ala Thr Thr Gly Lys Pro His Val Ala Tyr Phe Val 1 5 10
15Pro611PRTHomo sapiens 6Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr
Tyr 1 5 10710PRTHomo sapiens 7Ala Ser Ala Ala Tyr Pro Asp Pro Ser
Lys 1 5 10811PRTBos taurus 8Trp Asp Val Leu Glu Leu Arg Thr Ser Tyr
Tyr 1 5 10910PRTBos taurus 9Ser Ser Ala Ala Tyr Pro Asp Pro Ser Lys
1 5 101011PRTDrosophila melanogaster 10Trp Ser Leu Leu Glu Leu Arg
Thr Lys Tyr Tyr 1 5 101110PRTDrosophila melanogaster 11Ser Ala Ala
Ala Tyr Pro Pro Pro Ala Lys 1 5 101217PRTCaenorhabditis elegans
12Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro His Val Gly Tyr Leu Val 1
5 10 15Pro1311PRTCaenorhabditis elegans 13Trp Glu Leu Leu Lys Cys
Arg Val Ile Tyr Tyr 1 5 101410PRTCaenorhabditis elegans 14Lys Glu
Lys Gly Tyr Asn His Ser Thr Asp 1 5 101517PRTSaccharomyces
cerevisiae 15Tyr Trp Gly Thr Ala Pro Thr Gly Arg Pro His Cys Gly
Tyr Phe Val 1 5 10 15Pro1611PRTSaccharomyces cerevisiae 16Leu Glu
Val Val Asn Tyr Arg Ala Lys Tyr Tyr 1 5 101710PRTSaccharomyces
cerevisiae 17Ser Glu Lys Gly Tyr Pro Val Ala Thr Pro 1 5
101817PRTSaccharomyces pombe 18Tyr Trp Gly Ser Ala Pro Thr Gly Arg
Pro His Cys Gly Tyr Phe Val 1 5 10 15Pro1911PRTSaccharomyces pombe
19Met Glu Leu Val Gln His Arg Val Arg Tyr Tyr 1 5
102010PRTSaccharomyces pombe 20Leu Lys Ala Ala Tyr Pro Asp Pro Lys
Asp 1 5 102118PRTEscherichia coli 21Tyr Cys Gly Phe Asp Pro Thr Ala
Asp Ser Leu His Leu Gly His Leu 1 5 10 15Val Pro2211PRTEscherichia
coli 22Leu Asn Thr Glu Glu Thr Val Gln Glu Trp Val 1 5
10239PRTEscherichia coli 23Asp Gly Val Pro Met Val Glu Met Glu 1
52418PRTBacillus subtilis 24Tyr Ser Gly Phe Asp Pro Thr Ala Asp Ser
Leu His Ile Gly His Leu 1 5 10 15Leu Pro2511PRTBacillus subtilis
25Leu Asn Thr Ala Asp Ile Val Ser Glu Trp Ser 1 5 102610PRTBacillus
subtilis 26Asp Val Pro Ser Met Glu Val Asp Ser Thr 1 5 10
* * * * *